DNA replication generates pairs of sister chromatids that are held together until anaphase onset by a protein complex named cohesin. The phenomenon, termed sister chromatid cohesion, is critical for the proper loading of chromatids onto mitotic and meiotic spindles. In human cell lines, defects in the pathway produce levels of chromosome instability that match those seen in cancers. In humans, other pathway defects are responsible for at least three developmental diseases: Cornelia de Lange Syndrome (CdLS), Roberts syndrome (RBS) and SC phocomelia (SC). At the cellular level, RBS and SC are characterized by a striking loss of cohesion in chromatid domains containing heterochromatin, a form of chromatin that was first noted for its constitutively condensed appearance. Heterochromatin is now known to contain specialized histone modifications and non-histone binding proteins that hinder a variety of DNA transactions, including transcription of most genes. Using a combined approach of fluorescence microscopy and site-specific recombination, my lab discovered that cohesion of heterochromatin-like domains in yeast Saccharomyces cerevisiae is distinct from cohesion elsewhere in the genome. More recently we found that cohesion of the transcriptionally silenced HMR locus relies specifically on Sir2, an integral heterochromatin component with NAD-dependent histone deacetylase activity, and a tRNA gene that serves as a boundary to one heterochromatin domain. Our yeast model system provides an ideal opportunity to investigate the mechanisms of heterochromatic cohesion. We have developed a novel set of cytological assays to study cohesion at specific sites that will be used in combination with chromatin immunoprecipitation to address four questions. First, we will identify the features of the tRNA gene that facilitate cohesion and determine the step in the cohesion pathway that they act. Second, we will determine whether Sir2 can recruit cohesin directly and identify the protein surfaces and binding partners necessary for recruitment. Third, we will test whether typical components of the cohesin pathway contribute to the specialized form of cohesion at heterochromatin. We will also isolate heterochromatin fragments to examine the binding mode of cohesin in vitro. Last, we will use molecular genetic assays to assess the role of cohesion in heterochromatic silencing and genome stability.